1. Field of the Invention
This invention relates to the field of genetic engineering and is particularly related to the expression of proteins by techniques involving genetic engineering.
2. Description of the Background
The Renilla, also known as sea pansies, belong to a class of coelenterates known as the anthozoans. In addition to Renilla, other representative bioluminescent genera of the class Anthozoa include Cavarnularia, Ptilosarcus, Stylatula, Acanthoptilum, and Parazoanthus. All of these organisms are bioluminescent and emit light as a result of the action of an enzyme (luciferase) on a substrate (luciferin) under appropriate biological conditions. Prior studies have demonstrated that all of the above-mentioned anthozoans contain similar luciferases and luciferins. See, for example, Cormier et al., J. Cell. Physiol. (1973) 81: 291-298. The luciferases and luciferins from each of these anthozoans will crossreact with one another to produce the characteristic blue luminescence observed in Renilla extracts. Each of these luciferases has similar biochemical properties, and the biochemical requirements for bioluminescence are identical regardless of the anthozoan from which the luciferase was derived.
There has been considerable interest of late in replacing radioactive labels used in analytical assays with other types, such as luminescent labels. Firefly luciferase, which is a molecule of significantly different structure that does not react with Renilla-like luciferins, is one molecule that has been proposed for use as such labels. However, firefly luciferase suffers from a number of deficiencies that make this molecule less than optimal in biological assays. For example, ATP acts as a trigger of the firefly luciferase system, and the ubiquitous nature of ATP makes control of this variable difficult.
A prior patent application by one of the present inventors, U.S. patent application Ser. No. 059,137, filed Jun. 5, 1987, describes use of coelenterate-derived luciferases and photoproteins as bioluminescent labels. Other applications by the same inventor, for example, U.S. application Ser. Nos. 173,045, filed Mar. 17, 1988, and 165,422, filed Feb. 29, 1988, describe recombinant DNA capable of expressing the photoprotein apoaequorin.
The photoprotein aequorin (which consists of apoaequorin bound to a coelenterate luciferin molecule) and Renilla luciferase both utilize the same coelenterate luciferin, and the chemistry of light emission in both cases has been shown to be the same. However, aequorin luminescence is triggered by calcium, does not require dissolved oxygen, and represents a single turnover event. In contrast, Renilla luciferase is not triggered by calcium and requires dissolved oxygen in order to produce light in the presence of coelenterate luciferin. Renilla luciferase also acts as a true enzyme, catalyzing a long-lasting luminescence in the presence of saturating levels of luciferin.
Sub-attomole levels of aequorin can be detected with photometers even though its luminescence represents a single turnover event. Renilla luciferase, because of its enzymatic ability, should be detectable at levels 1 to 2 orders of magnitude lower than aequorin. Furthermore, Renilla luciferase is known to be relatively stable to heat, an important consideration for assays that often involve incubation at physiological temperatures. Accordingly, Renilla luciferase is a potentially useful label for biological and other assays.
On the other hand, Renilla live on the ocean bottom, about 30 to 100 feet deep, and must be collected by dregging. From 1 kg of Renilla (about 1000 animals), approximately 1 mg of pure Renilla luciferase can be obtained following a tedious procedure which requires purifying the protein about 12,000 fold. The purification procedure is described in Matthews et al., Biochemistry (1977) 16: 85-91. As a result, there has been no development of Renilla luciferase as a detectable label.
Accordingly, improved techniques for the production of pure Renilla luciferase are necessary before this molecule can be used commercially in bioluminescence assays.